A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

Dong-A University

Akt/PKB plays a pivotal role in cell proliferation and survival. However, the isotype-specific roles of Akt in mitochondrial function have not been fully addressed. In this study, we explored the role of Akt in mitochondrial function after stable knockdown of the Akt isoforms in EJ human bladder cancer cells. We found that the mitochondrial mass was significantly increased in the Akt1- and Akt3-knockdown cells, and this increase was accompanied by an increase in TFAM and NRF1. Akt2 knockdown did not cause a similar effect. Interestingly, Akt3 knockdown also led to severe structural defects in the mitochondria, an increase in doxorubicin-induced senescence, and impairment of cell proliferation in galactose medium. Consistent with these observations, the mitochondrial oxygen consumption rate was significantly reduced in the Akt3-knockdown cells. An Akt3 deficiency-induced decrease in mitochondrial respiration was also observed in A549 lung cancer cells. Collectively, these results suggest that the Akt isoforms play distinct roles in mitochondrial function and that Akt3 is critical for proper mitochondrial respiration in human cancer cells. 

Akt3 knockdown induces mitochondrial dysfunction in human cancer cells.

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagic machinery. Mitophagy plays an essential role in maintaining mitochondrial quality control and homeostasis. Mitochondrial dysfunctions and defective mitophagy in neurodegenerative diseases, cancer, and metabolic diseases indicate a close link between human disease and mitophagy. Furthermore, recent studies showing the involvement of mitophagy in differentiation and development, suggest that mitophagy may play a more active role in controlling cellular functions. A better understanding of mitophagy will provide insights about human disease and offer novel chance for treatment. This review mainly focuses on the recent implications for mitophagy in human diseases and normal physiology. [BMB Reports 2017; 50(6): 299-307].

Emerging role of mitophagy in human diseases and physiology.

Cellular senescence has been implicated in normal aging, tissue homeostasis, and tumor suppression. Although p53 has been shown to be a central mediator of cellular senescence, the signaling pathway by which it induces senescence remains incompletely understood. In this study, we have shown that both Akt and p21 are required to induce cellular senescence in response to p53 expression. In a p53-induced senescence model, we found that Akt activation was essential for inducing a cellular senescence phenotype. Surprisingly, Akt inhibition did not abolish p53-induced cell cycle arrest, but it suppressed the increase in intracellular reactive oxygen species (ROS) levels. The results of the cell cycle and morphological analysis suggest that p53 induced quiescence, not senescence, following Akt inhibition. Conversely, the inhibition of p21 induction abolished cell cycle arrest but did not affect the p53-induced increase in ROS levels. Additionally, p21 and Akt separately controlled cell cycle arrest and ROS levels, respectively, during H-Ras-induced senescence in human normal fibroblasts. The mechanistic analysis revealed that Akt increased ROS levels through NOX4 induction, and increased Akt-dependent NF-κB binding to the NOX4 promoter is responsible for NOX4 induction upon p53 expression. We further showed that Akt activation upon p53 expression is mediated by mammalian target of rapamycin complex 2. In addition, p53-mediated IL6 and IL8 induction was abrogated by Akt inhibition, suggesting that Akt activation is also required for the senescence-associated secretory phenotype. Collectively, these results suggest that p53 simultaneously controls multiple pathways to induce cellular senescence through p21 and Akt.

Cooperation between p21 and Akt is required for p53-dependent cellular senescence.

MicroRNAs are small non-coding RNAs that bind to the 3'-untranslated region of target mRNAs and have transcriptional or translational inhibitory function in eukaryotes. Before microRNAs were widely known, bacterial non-coding small RNAs around 50-200 nt in length were discovered whose mechanism of action resembled that of microRNAs. Recently, RNAs that are of similar size to or smaller than microRNAs have been discovered in bacteria and indeed, this class of small RNAs have been found throughout all domains of life. Moreover, recent findings suggest that these tiny RNAs can be released via extracellular vesicles (such as exosomes in eukaryotes and outer membrane vesicles in bacteria), which in turn heralds a new field of research, interkingdom communication. This review discusses two similar classes of small RNAs in evolutionarily distinct eukaryotes and bacteria. In addition to their biogenesis and regulation, we discuss small RNA vehicles and their secretion. Impact statement The possible endogenous functions of small RNAs such as regulatory small RNAs in bacteria and microRNAs in eukaryotes have been extensively studied since they were first discovered. However, their powerful functions should not be seen as limited to their cells of origin. Recently, several papers have demonstrated that small RNAs function as signaling molecules between cells. This is possible because small RNAs can be shuttled around after being incorporated into environmentally protective extracellular vesicles. It is now clearly plausible that secreted small RNAs can regulate other types of cells through biofluids. Given their "common molecule" status, the role of small RNAs in mediating bacteria-human crosstalk is an emerging and competitive area of genetic research. This review provides insight into the function of small RNAs in intercellular and even interkingdom communication.

Tiny RNAs and their voyage via extracellular vesicles: Secretion of bacterial small RNA and eukaryotic microRNA.

Activation of adiponectin receptors (AdipoRs) by its natural ligand, adiponectin has been known to be involved in modulating critical metabolic processes such as glucose metabolism and fatty acid oxidation as demonstrated by a number of in vitro and in vivo studies over last two decades. These findings suggest that AdipoRs' agonists could be developed into a potential therapeutic agent for metabolic diseases, such as diabetes mellitus, especially for type II diabetes, a long-term metabolic disorder characterized by high blood sugar, insulin resistance, and relative lack of insulin. Because of limitations in production of biologically active adiponectin, adiponectin-mimetic AdipoRs' agonists have been suggested as alternative ways to expand the opportunity to develop anti-diabetic agents. Based on crystal structure of AdipoR1, we designed AdipoR1's peptide agonists using protein-peptide docking simulation and screened their receptor binding abilities and biological functions via surface plasmon resonance (SPR) and biological analysis. Three candidate peptides, BHD1028, BHD43, and BHD44 were selected and confirmed to activate AdipoR1-mediated signal pathways. In order to enhance the stability and solubility of peptide agonists, candidate peptides were PEGylated. PEGylated BHD1028 exhibited its biological activity at nano-molar concentration and could be a potential therapeutic agent for the treatment of diabetes. Also, SPR and virtual screening techniques utilized in this study may potentially be applied to other peptide-drug screening processes against membrane receptor proteins.

Jee-Hyun Um

Department of Biochemistry,
College of Medicine,
Peripheral Neuropathy Research Center

Dong-A University

A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Jee-Hyun Um

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry,College of Medicine,Peripheral Neuropathy Research Center

Dong-A University

A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Department of Biochemistry,
College of Medicine,
Peripheral Neuropathy Research Center